Umbilical cord (UC) tissue has been established as a reliable source of stem cells for successful hematopoietic stem cell transplantations (HSCT). More than 5000 allogeneic cord blood (UCB) transplantations have been reported to treat both adults and children with hematologic diseases (1-5). Success can be attributed to several advantages including 1) accessibility and ease of cell procurement, 2) absence of risks for donors, 3) reduced likelihood of transmitting infections and 4) ability to store fully tested and HLA-typed transplants (1). The readily available source of human umbilical cord tissue and the current clinical success of cord blood transplantation for hematological disorders suggest an expedited route for translation of other UC-derived cells. Also, as an allogeneic transplantation, UCB cells are more histocompatible than adult-derived cells, require fewer HLA matches and report reduced graft-versus-host disease. There is a reduced likelihood of infectious disease contamination. While the most intensely investigated UC-derived stem cells have come from the blood and have been used clinically for blood disorders, new investigations are revealing that additional cells derived from the perivascular region, which is closely associated with the vasculature, can differentiate into cells of the cardiomyogenic, adipogenic, osteogenic, and neural lineages (6- 14). Wharton's jelly (WJ) surrounds the cord vessels and consists of proteoglycans, collagen, hyaluronic acid and fibroblast-like cells. Mainly, these WJ cells have been characterized by microscopy and histologic techniques (15, 16);more recently we have described the presence of cells with stem cell characteristics in the WJ. We have developed methods to isolate phenotypically-defined, WJ stem cells to a yield not previously reported. To date, we have found that our methods can obtain 3.5 x 105 cells per gram of digested tissue. We also determined that the UC may contain, by conservative estimate, approximately 12 million cells per gram of tissue, and the average cord is 40 grams. This yield is higher than previous reports using different isolation and processing methods (11, 17), and perhaps isolating different cells. Further optimization is possible since the entire cord contains a theoretical yield of 500 million cells. These cells have a mesenchymal stem cell (MSC) phenotype and differentiate to musculoskeletal lineages. In this study, we propose specific aims to further develop the isolation procedures to increase yield and optimize cell culture of these novel populations of stem cells (Aim1). We also propose to confirm the viability and stability of the stem cell phenotype after processing, cryopreservation and thawing (Aim 2). The overall goal of the Phase I/Phase II project is to develop a UC cell product that can be used allogeneically in off-the-shelf products for a number of regenerative medicine applications. Here, we will demonstrate the product compliance with the FDA's regulatory framework for the collection, processing, storage, labeling, packaging and distribution of human cells, as outlined by the current cellular Good Tissue Practices (cGTP) and cellular Good Manufacturing Practices (cGMP) standards in 21 CFR Parts 1270-71. PUBLIC HEALTH RELEVANCE: As a readily available and unlimited resource, stem cells isolated from the human umbilical cord have the potential to revolutionize the field of regenerative medicine and tissue engineering. These cells have potential to be used for un-related donors to treat diseases related to cartilage, bone, muscle or tendons/ligaments. By maximizing the cell yield from each cord and developing techniques to store these cells for extended periods, we can develop a large stockpile of ready-to- use replacement cells fit for the entire population. This project will study how to maximize the stocks of these cells and ensure their safe use in cell therapy.